Mechanism and Substrate Specificity of the Flavin Reductase ActVB from Streptomyces coelicolor

• Published in: The Journal of biological chemistry 2003-01, Vol.278 (1), p.296-303
• Main Authors: Filisetti, Laurent, Fontecave, Marc, Nivière, Vincent
• Format: Article
• Language: English
• Subjects: Binding Sites; Biochemistry; Biochemistry, Molecular Biology; Flavin Mononucleotide - chemistry; Flavin Mononucleotide - metabolism; Flavins - chemistry; Flavins - metabolism; FMN Reductase - metabolism; Kinetics; Life Sciences; Molecular Structure; NAD - chemistry; NAD - metabolism; Nucleotides - chemistry; Oxidation-Reduction; Streptomyces - enzymology; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M209689200

ActVB is the NADH:flavin oxidoreductase participating in the last step of actinorhodin synthesis inStreptomyces coelicolor. It is the prototype of a whole class of flavin reductases with both sequence and functional similarities. The mechanism of reduction of free flavins by ActVB has been studied. Although ActVB was isolated with FMN bound, we have demonstrated that it is not a flavoprotein. Instead, ActVB contains only one flavin binding site, suitable for the flavin reductase activity and with a high affinity for FMN. In addition, ActVB proceeds by an ordered sequential mechanism, where NADH is the first substrate. Whereas ActVB is highly specific for NADH, it is able to catalyze the reduction of a great variety of natural and synthetic flavins, but withKm values ranging from 1 μm (FMN) to 69 μm (lumiflavin). We show that both the ribitol-phosphate chain and the isoalloxazine ring contribute to the protein-flavin interaction. Such properties are unique and set the ActVB family apart from the well characterized Fre flavin reductase family.